?-agonists acting at the ?2-adrenergic receptor (?2AR) on human airway smooth muscle (HASM) relax the muscle and thus dilate the airways, and are used for acute (rescue) and chronic (maintenance) therapy for asthma. With many asthmatics not achieving control, there is a need to understand the loss of efficacy observed with acute -agonists and the development of tolerance, or tachyphylaxis, during chronic therapy. These issues are due in part to a lack of understanding of fundamental aspects of ?2AR signaling in HASM. This proposal has three aims to close this gap, with the broad, long-term objective of developing optimal -agonist treatment for asthma to reduce morbidity. In Aim 1, we will screen a 40 million compound library to discover -agonists that stabilize a specific ?2AR conformation that is favorable for asthma. This biasing would be towards Gs/cAMP coupling (improves bronchodilation) and away from -arrestin recruitment (thus minimizing tolerance). This will be accomplished through a sequential screening approach that measures cAMP, then -arrestin recruitment, and then physiologic function in human airways. The compounds that come from this screening will be candidates for preclinical trials in asthma. Moreover, their structures will teach us the molecular basis for engineering agonists to exert specific functions from receptors. In Aim 2, the interactions between ?2AR and another airway receptor that bronchodilates, the bitter taste receptor (TAS2R), will be ascertained. Agonists for TAS2Rs are in development for treating asthma, and it is envisioned that TAS2R and ?2AR agonists will be administered concomitantly. Yet, the two receptors appear to be intertwined at the cell surface in heterodimers, where activation of one receptor alters function of the other. Heterodimers and their function will be studied using bimolecular fluorescent complementation, co-immunoprecipitation, biotinylation, and confocal microscopy. Function will be ascertained by measuring the second messengers from each monomeric component (Ca2+ for TAS2R; cAMP for ?2AR). In Aim 3, the mechanism of translational repression of the ?2AR gene by miRNAs will define how the resting level of ?2AR protein is established on HASM. This level is also a determinant of the acute response to -agonist, and miRNAs themselves appear to be modulated by agonists. These events will be studied with selected members of the let7, miR15, and miR30 miRNA families by overexpression, knock-downs, and gene and protein expression measurements in model cells and HASM. Collectively, these studies will define mechanisms that point to new therapeutics within the -agonist pathway for improved therapy of asthma.